1-dehydro-11-keto progesterone



l-DEHYDRO-ll-KETO PROGESTERONE Samuel H. Eppstein, Galesburg, and PeterD. Meister, Kalamazoo Township, Kalamazoo County, Mich., and AdolphWeiutrauh, Brooklyn, N.Y., assignors to The Upjohn Company, Kalamazoo,Mich., a corporation of Michigan No Drawing. Application November 2,1956 Serial No. 619,952

1 Claim. (Cl. 260-3975) The invention relates to a new chemicalcompound, 1- dehydro-l l-ketoprogesterone (1,4 pregnadiene-3,11,20-trione), which compound possesses valuable glucocorticoid andanti-inflammatory activity.

. This application is a continuation-in-part of application S.N.493,302, filed March 9, 1955, now abandoned.

The compound of this invention has the following structural formula:

The compound is useful also as a starting material for the preparationof l-dehydrohydrocortisone and l-dehydrocortisone, using onlymicrobiological conversion steps. For example, it is readily convertibleby the method disclosed by Ch. Meystre, E. Vischer, and A. Wettstein,Helv. Chim. Acta, 37, 1548 (1954) to the 21-hydroxylated product, 21hydroxy 1,4 pregnadiene 3,11,20- trione, i.e., by fermentation with21-hydroxylated microorganisms such as Ophiobolus herpotrichus. Thislatter compound is readily converted, without isolation from theorganism-free beer if desired, to l-dehydrocortisone by the process ofP. D. Meister et al., I. Am. Chem. Soc., 76, 4050 (1954), i.e., byfermentation with a 17a-hydroxylating microorganism such asCephalothecium r0- .reum. This latter compound and its 21-acylatespossess known valuable therapeutic properties. The 21-acy1ates,moreover, are convertible to l-dehydrohydrocortisone 21- acylates byknown techniques, i.e., by reduction with lithium aluminum hydride orsodium borohydride. The latter compounds and the corresponding freealcohol, i.e., l-dehydrohydrocortisone, possess known valuabletherapeutic properties.

As disclosed above, the compound of this invention possesses valuableglucocorticoid and anti-inflammatory activity. Its pharmacologicalproperties are especially superior to those possessed byll-ketoprogesterone, a parent compound from which it can be made asdisclosed below.

The novel compound of this invention can be produced by a fermentativel-dehydrogenation of ll-ketoprogesterone with a fungus of the genusSeptomyxa under aerobic conditions as disclosed in the copending priorapplication previously referred to.

As disclosed in the prior application, the operational conditions andreaction procedure and details of production can be those already knownin the art of steroid bioconversion as illustrated by US. Patent2,602,769, uti1iz ing however the action of a species of fungus of thegenus United States Patent 0 2,883,400 Patented Apr. 21, 1959 Septomyxa.The genus Septomyxa belongs to the class of Deuteromyces, Fungiimperfecti, of the order Melanooniales, of the family Melanconiaceae.Among the species of the genus Septomyxa which are useful in theconversion of ll-ketoprogesterone to l-dehydro-ll-ketoprogesterone areSeptomyxa afiinis (Sherb.) Wr., A.T.C.C. 6737, American Type CultureCollection, 2029 M Street, N.W., Washington 6, DC, Septomyxa aesculi,Septomyxa corni, Septomyxa salicina, and Septomyxa tulasnei.

As disclosed in the prior application, culture of the fungi for theproduction of l-dehydroprogesterone is in or on a medium favorable tothe development of the fungi, employing conventional sources ofassimilable carbon and assimilable nitrogen; and using conventionalsources of mineral nutrients.

The compound of this invention can also be prepared by oxidizing1la-hydroxy-l-dehydroprogesterone with chromic acid. The latter compoundcan be prepared by the fermentative l-dehydrogenation ofllot-hydroxyprogesterone with a fungus of the genus Septomyxa underaerobic conditions as described above in connection with thel-dehydrogenation of ll-ketoprogesterone.

The following preparation and examples are illustrative of theproduction of the novel compound, and are not to be construed aslimiting.

PREPARATION 1 1 a-hydroxy-I -dehydr0progester0ne 25 liters of a mediumcontaining one percent commercial dextrose hydrate, one percentcornsteep liquor (sixty percent solids) and tap water was adjusted to pH5.1 by adding 49 milliliters of 25 percent sodium hydroxide solution.The entire tank system containing the medium was sterilized for a periodof 35 minutes at 120 degrees centigrade. The medium was allowed to cool,and air was introduced through a sparger at the rate of 0.25 liter perminute, and the medium was agitated mechanically.

After the temperature of the medium was adjusted to between 25 degreesand 28 degrees centigrade, in0cu1ation was carried out by introducing anisotonic saline suspension of Septomyxa afiinis spores. The medium wascultured for 25 hours, at which time 5.0 grams of hydroxyprogesteronedissolved in 250 milliliters of dipropylene glycol was added, andfermentation was allowed to progress for an additional 25 hours.

At the end of the fermentation period, the beer was extracted four timeswith six liters of methylene chloride, and the extract washed once withwater. The water wash was extracted with one liter of methylene chloridewhich was combined with the original extract, and the whole was driedover anhydrous sodium sulfate overnight. Papergram analysis of analiquot sample indicated that all steroidal material contained in theextract was the desired product, namely,11a-hydroxy-l-dehydroprogesterone.

The extract was evaporated to dryness and the residue triturated oncewith 25 milliliters of ether to give 3.68 grams of crude crystals (73percent yield), M.P. 227 to 230 degrees centigrade. 3.36 grams of thiscrystalline product was recrystallized twice from methanol to give anoverall yield of fifty percent of pure crystals of melting point 232.5degrees to 234 degrees centigrade, [041 plus 117 degrees in chloroform(1.3186 conc.),

Infrared spectrum indicates absorption as follows: OH, 3420 ems-20-ketone, 1699 cmr conjugated ketone, 1655 cm.- A -structure ((#C),1618, 1596 cmr- Analysis.-Calculated for C H O C, 76.79; H. 8.59. Found:C, 77.10; H. 8.64.

EXAMPLE 1 1 -dehydr-1 1 -ket0 progesterone One gram of11a-hydroxy-l-dehydroprogesterone, as produced in the foregoingpreparation, was oxidized by dissolving in thirty milliliters of aceticacid containing 300 milligrams of chrominum trioxide and a drop ofwater. The reaction mixture was allowed to stand at room temperatureovernight, after which time ten milliliters of methanol was added, thereaction mixture filtered, and the filtrate evaporated to dryness in anair stream. The residue, which was crystalline, was extracted withmethylene chloride, the extract washed with two percent sodium carbonatesolution which was followed by a water wash and the washed filtratedried with anhydrous sodium sulfate. The extract was evaporated todryness and the residue crystallized from acetone. The crystals,l-dehydro-ll-ketoprogesterone, melted at 178 to 179.5 degreescentigrade. After two recrystallizations from acetone the melting pointwas 179 to 181 degrees centigrade.

Elementary analysis, ultraviolet, and infrared spectral analysis andoptical rotation were in agreement with these constants obtained for theidentical product pro duced in Example 2, below.

EXAMPLE 2 1 -dehydro-1 1 -ketoprogester0ne Twelve liters of a medium ofthe following composition:

Percent by weight Commercial dextrose 1 Cornsteep liquor (sixty percentsolids) 2 was adjusted to pH 4.9 in a five-gallon stainless steelfermentor and autoclaved for one hour at fifteen pounds steam pressure.The medium was cooled and then inoculated with 600 milliliters of a24-hour culture of Septomyxa afiinis A.T.C.C. 6737 (grown from spores inthe same type of medium). Air was sparged at the rate of 0.1 liter perminute and the medium was stirred at 200 rpm. After the culture hadgrown for seventeen hours, three grams of ll-ketoprogesterone dissolvedin 75 milliliters of acetone was added, and the fermentation allowed toproceed for an additional 24 hours.

At the end of the 24-hour fermentation period, the contents of thefermentor were filtered. The filtered beer and the separated myceliumwere extracted with methylene chloride and the combined extracts washedwith two percent sodium bicarbonate solution followed by a water wash.The washed extract was dried with anhydrous sodium sulfate andevaporated to an oily, semicrystalline residue.

The residue was triturated with Skellysolve B hexanes. The gummy residuewas taken up in ten milliliters of boiling ethyl acetate. The ethylacetate solution was cooled to room temperature (twenty to 25 degreesCentigrade), and crystallized initially by scratching the side of thecontainer with a stirring rod. The crystals were filtered and thematerial recrystallized twice from ethyl acetate yielding purel-dehydro-ll-ketoprogesterone in a total yield of thirty percent basedon the ll-ketoprogesterone substrate. The compound had the followingcharacteristics: M.P. 179 to 181 degrees centigrade, [u] =224 (in CHCl 00.838),

x33, 240 m K=45.69, 6 14,925 Infrared spectrum indicates absorption asfollows: ketone, 1700 cmconjugated ketone, 1665 CH1. 1; A -diene, 1626cm.- and 1604 cm.-

Analysis-Calculated for C H O C, 77.27; H, 8.03. Found: C, 77.50; H,8.51.

l-dehydro-ll-ketoprogesterone of this invention exexhibits valuableglucocorticoid and anti-inflammatory activity and can be administered inthe form of tablets, capsules, syrups and the like for oral use, insuitable suspension media for injection use and in ointments, lotionsand cremes for topical use.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds shown and described, asobvious modifications and equivalents will be apparent to one skilled inthe art, and the invention is therefore to be limited only by the scopeof the appended claims.

We claim:

1,4-pregnadiene-3,11,20-trione.

References Cited in the file of this patent UNITED STATES PATENTSI-Ianze Apr. 20, 1954 Fried et a1. July 24, 1956 Nobile et al.:J.A.C.S., vol. 77, page 4184, August 5, 1955.

